Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee - PubMed (original) (raw)

Comparative Study

Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee

Charles W Whitfield et al. Genome Res. 2002 Apr.

Abstract

To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708-BI517278. The sequences are also available at http://titan.biotec.uiuc.edu/bee/honeybee\_project.htm.\]

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Figures

Figure 1

Figure 1

Open reading frame (ORF) and

BLASTX

results. (A) The proportion of assembled sequences with and without

BLASTX

matches in the Non-Redundant Protein (nr) database (E ≤10−5) is indicated for assembled sequences with and without an identified 450 bp ORF. Relative area of pie charts indicates number of sequences. (B) Apis sequences with matches in the nr database (3501 total) were classified by the organism of the “best hit” protein sequence. (C) Apis sequences with matches in the nr database (3501 total) were separately analyzed for matches in Arthropoda and Chordata protein databases (see Table 8 for sub-database creation).

Figure 2

Figure 2

Signal intensities from an example microarray. Values plotted are feature minus background intensity at 635 and 532 nm wavelengths for each cDNA spot (see Methods). Values were normalized such that the median ratio (635:532 nm) equals 1.0. Apis cDNAs are shown as black x's, exogenous negative control cDNAs are shown as red x's. Cy3-labeled probe (532 nm) and Cy5-labeled probe (635 nm) were independently derived from the same starting sample (using in vitro transcription to amplify starting mRNA; see Methods). The starting sample consisted of a mixture of two dissected adult bee brains (one bee observed foraging and one bee observed caring for brood). The coefficient of correlation (r) between 635 and 532 nm values was 0.9926 (based on log-transformed values). Divergence of values from the diagonal (ratio = 1) reflects technical variation introduced during RNA isolation, mRNA amplification by in vitro transcription, and fluorescent labeling of probe. The two diagonal bars indicate ratios (635:532 nm) equal to 0.5 and 2.0.

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